EP0316492B1 - Polymeric matrix for affinity chromatography and immobilization of ligands - Google Patents

Polymeric matrix for affinity chromatography and immobilization of ligands Download PDF

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EP0316492B1
EP0316492B1 EP87310279A EP87310279A EP0316492B1 EP 0316492 B1 EP0316492 B1 EP 0316492B1 EP 87310279 A EP87310279 A EP 87310279A EP 87310279 A EP87310279 A EP 87310279A EP 0316492 B1 EP0316492 B1 EP 0316492B1
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process according
polymer
polyethyleneimine
gel
polymeric substance
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EP0316492A1 (en
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That T. Ngo
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Unisyn Fibertec Corp Te San Diego Californ
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Bioprobe International Inc
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    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
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Definitions

  • This invention relates to affinity chromatography.
  • this invention relates to a matrix for use in affinity chromatography and in the immobilization of ligands.
  • biologically active materials can be purified by a process which involves immobilization of the biologically active material, which will be referred to herein as a ligand, upon a suitable polymeric matrix, followed by separation of the immobilized ligand from the mixture in which it is present.
  • the ligand can then be used in its immobilized form, if desired, or it can be released from the matrix on which it is immobilized by suitable chemical treatment and used in its non-immobilized form.
  • One of the first methods for immobilizing biological ligands involved treatment of a polymer containing hydroxyl groups with an activating agent such as cyanogen bromide, CNBr.
  • the activated polymer could then be used to directly bind various biological ligands to the polymer by means of covalent bonds.
  • Porath et al. describe several chemical activation methods, including the CNBr method, in Porath, et al., "Immobilized Enzymes", Methods in Enzymology , K. Mosbach, Ed., Vol. 44, pp. 19-45, Academic Press, New York (1976).
  • CNBr activation procedures suffer from the following disadvantages: (1) the linkages formed between CNBr-activated hydroxyl containing polymers and the amino groups of ligands which are reacted with the activated polymers are labile; (2) the reaction between the activated polymer and ligand frequently results in the introduction of charged species which interfere with utilization of the reaction product in affinity absorption; and (3) CNBr is a noxious lachrimatory and poisonous chemical which requires special care in its handling.
  • a spacer that is, an organic compound, which can be placed between the polymer surface and the ligand, helps to overcome the steric hindrance due to close proximity of the bound ligand to the polymer matrix.
  • Both straight chain spacers and branched chain spacers have been used for this purpose.
  • P.V. Sundaram "Potentials of Enzymes Attached to Nylon Tubes in Analysis," Biomedical Applications of Immobilized Enzymes and Proteins , Vol. 2, T.M.S. Chang, Ed., pp. 317-340, Plenum Press, New York (1977), use of a poly-lysine spacer is illustrated (at page 323).
  • pectinamine a polyamine derived from 1,3-diaminopropan-2-ol and pectin, for the covalent attachment of enzymes, is shown (at page 271).
  • U.S. Patent No. 4,152,411 describes the preparation and use of a marked spine diagnostic tool, for example, one containing thyroxine, poly-l-lysine and horseradish peroxidase.
  • the present invention provides a matrix useful in affinity chromatography and ligand immobilization and a process for preparing the same which involves the use of a polymeric non-biologically active spacer.
  • the spacer used is polyethyleneimine.
  • the matrix of the present invention is prepared by a process which includes the steps of reacting a hydroxyl containing polymer with 2-fluoro-1-methylpyridinium toluene-4-sulfonate (FMP) to form an activated polymer wherein at least some of the hydroxyl groups of the polymer have been substituted to form 1-methyl-2-pyridoxy toluene-4-sulfonate groups, and reacting the 1-methyl-2-pyridoxy substituted polymer with polyethyleneimine to form an affinity chromatographic matrix in which at least some of the 1-methyl-2-pyridoxy groups of the polymer have been replaced by polyethyleneimine.
  • FMP 2-fluoro-1-methylpyridinium toluene-4-sulfonate
  • the present invention provides the means for immobilizing and covalently chromatographing a wide variety of organic ligands, such as enzymes, nucleoproteins, antigens, antibodies, haptens, hormones, vitamins, polypeptides and other biologically active compounds.
  • Ligands having biological activity may under ordinary circumstances be subject to loss of biological activity due to steric interactions with chromatographic matrices. Use of a polyethyleneimine spacer can prevent such loss.
  • Ligands which contain electrophilic groups capable of reacting with a polyethyleneimine can be readily chromatographed by this method.
  • Such ligands include acylating agents such as carboxylic acids, acyl halides, sulfonyl halides, N-hydroxysuccinimides, active esters such as p-nitrobenzoates, imidoesters, aldehydes, epoxides, thioepoxides, divinylsulfones and the like.
  • acylating agents such as carboxylic acids, acyl halides, sulfonyl halides, N-hydroxysuccinimides, active esters such as p-nitrobenzoates, imidoesters, aldehydes, epoxides, thioepoxides, divinylsulfones and the like.
  • active esters such as p-nitrobenzoates, imidoesters, aldehydes, epoxides, thioepoxides, divinylsulfones and the like.
  • the first step in the process is the reaction of a hydroxyl containing polymer having at least one reactive hydroxyl group (Formula 1) with 2-fluoro-1-methylpyridinium toluene-4-sulfonate (Formula 2), as shown in Figure 1.
  • polymeric materials can be used as long as they contain at least one reactive hydroxyl group.
  • Principal among the polymers which may be used are the polysaccharides, such as the celluloses, including paper; dextrans and cross-linked dextrans; and agaroses and cross-linked agaroses.
  • Other polymeric materials which can be used include natural, semi-synthetic and synthetic materials containing hydroxyl groups such as polyethylene glycols; polyvinyl alcohols, for example, FRACTOGEL TSK, a porous, semi-rigid spherical gel synthesized from hydrophilic vinyl polymer and composed exclusively of C, H and O atoms, supplied by E.
  • polyacrylates such as polyhydroxymethyl methyl acrylates or TRISACRYL GF 2000, a polymer of N-acryloyl-2-amino-2-hydroxymethyl-1,3-propanediol, manufactured by ReRET IBF, France; glycophase glass and silica particles having bonded groups containing at least one hydroxyl group bonded to a carbon atom.
  • the polymer will sometimes be referred to as a gel.
  • the polymer may be provided in the form of beads, if desired, or in any other convenient form.
  • 2-halo-1 methylpyridinium salts than FMP, such as 2-chloro-1-methylpyridinium salts, can be used, but the 2-fluoro-1-methylpyridinium salts are preferred because of their greater reactivity.
  • the reaction between the hydroxyl containing polymer and FMP or other 2-halo-1-methylpyridinium salt can be conducted in an aqueous solution or any polar organic solvent such as acetonitrile, acetone, dimethyl formamide or tetrahydrofuran in the presence of a base such as tertiary amine, for example, triethylamine (TEA) or tributylamine (TBA).
  • a base such as tertiary amine, for example, triethylamine (TEA) or tributylamine (TBA).
  • TAA triethylamine
  • TSA tributylamine
  • the activated polymer is readily attacked by nucleophiles, such as polyethylenemine (Formula 4), since the 1-methyl-2-pyridoxy group is a good leaving group, being readily converted to 1-methyl-2-pyridone (Formula 6) upon nucleophilic substitution.
  • nucleophiles such as polyethylenemine (Formula 4)
  • Form 4 polyethylenemine
  • 1-methyl-2-pyridoxy group is a good leaving group, being readily converted to 1-methyl-2-pyridone (Formula 6) upon nucleophilic substitution.
  • the reaction of the 2-alkoxy-1-methylpyridinium salt (Formula 3) with polyethyleneimine (Formula 4) is illustrated in Figure 1.
  • Polyethyleneimines varying in molecular weight from about 600 to 100,000 can be used. Molecular weights in the range of about 1,000 to 20,000 are preferred. Polyethyleneimines also vary with respect to the degree of branching. As the degree of branching increases, the number of amino groups available for further reaction increases. In Formulae 4 and 5 only one such branch is shown.
  • the reaction is conducted in a suitable polar organic solvent such as acetonitrile, acetone or tetrahydrofuran.
  • the resulting product contains a terminal amino group and will sometimes be referred to herein as an amino-terminal gel.
  • the unmodified amino-terminal gel can serve as an affinity chromatographic matrix for such electrophilic ligand.
  • the ligand of interest does not contain groups which are readily reacted with an amino group of the polyethyleneimine, for example, where the ligand itself contains reactive amino groups, it may be desirable to further derivatize the amino-terminal gel, by reaction with a suitable derivatizing agent or electrophilic ligand, to provide groups on the gel which can be readily reacted with the active functional groups of the ligand of interest as shown in Figure 2.
  • the amino-terminal gel for example, can readily be converted to a thiol gel (Formula 8) by reaction with ethylene sulfide (Formula 7).
  • the thiol gel can be activated by reaction with 2,2-dithiopyridine (Formula 13) to produce an activated thiol gel (Formula 14) which can be used as a chromatographic matrix for sulfhydryl containing ligands, such as sulfhydryl containing enzymes.
  • reaction of the derivatizing agent with the amino-terminal gel may take place at any available primary or secondary amino group.
  • SEPHAROSE CL-4B a cross-linked agarose gel from Pharmacia Fine Chemicals, Uppsala, Sweden, (5 ml.) was washed five times with 200 ml. distilled water each time.
  • the washed gel was suspended in 5 ml. distilled water which contained 1.2 mmoles 4-dimethylaminopyridine.
  • the gel suspension was stirred at room temperature for 5 minutes.
  • To this suspension was then added 1.0 mmole FMP.
  • the gel was continuously stirred for another 15 minutes and then washed with 200 ml. distilled water five times, with 200 ml. 1M NaCl five times and with 200 ml. distilled water five times.
  • TRISACRYL GF 2000 gel (1200 ml.) was washed successively with: 5 ⁇ 1600 ml. distilled water; 3 ⁇ 1600 ml. ethyl alcohol; and 1600 ml. dry acetone. After removing excess acetone by vacuum filtration, the gel was suspended in 1600 ml. dry acetone and stirred for 10 minutes. The gel was washed one more time with 1600 ml. dry acetone and excess solvent was removed by vacuum filtration. The dry gel was first placed in a drying oven at 80 degrees Celsius for 5 hours and then in 80 degrees Celsius vacuum oven for 14 hours. The dry gel was suspended in 500 ml. dry acetone containing 2.6 ml.
  • TEA dry triethylamine
  • DMF dry N,N-dimethyl formamide
  • the gel was washed with 1 l. dry DMF and suspended in 1 l. dry DMF with stirring for 10 minutes.
  • the FMP-activated gel was then washed successively with: 2 ⁇ 1000 ml. dry acetone; 1000 ml. acetone containing 4 mM HCl; 2 ⁇ 1000 ml. 4mM HCl; and 2 ⁇ 1000 ml. dry acetone.
  • the washed FMP-activated gel was dried at room temperature under vacuum.
  • Example 4 Activation of Filter Paper with 2-Fluoro-1-methylpyridinium Toluene-4-sulfonate (FMP) in Dimethyl Formamide (DMF) .
  • FMP 2-Fluoro-1-methylpyridinium Toluene-4-sulfonate
  • DMF Dimethyl Formamide
  • the dish was shaken at 100 rpm for 2 hours at room temperature. After removing the solution, 250 ml. dry DMF was added, and the dish was shaken at 100 rpm for 10 minutes. This washing procedure was repeated 2 more times. After decanting the DMF, 250 ml. dry acetone was added to the papers in the dish and the dish was shaken at 100 rpm for 10 minutes. This process of washing with dry acetone was repeated 2 more times. Finally, the papers were air-dried at room temperature and then stored desiccated at 4 degrees Celsius.
  • a quantity of 20 g. of amino-terminal gel in its dry form prepared according to the procedure of Example 5 as suspended in 120 ml. of dry acetone. To this suspension was added 4 ml. ethylene sulfide. The resulting mixture was stirred at room temperature for 18 hours. The gel was then washed successively with: 3 ⁇ 1000 ml. distilled water; 3 ⁇ 600 ml. acetone; 3 ⁇ 1000 ml. distilled water; 3 ⁇ 1000 ml. acetone; 3 ⁇ 1000 ml. 1 M NaCl; 3 ⁇ 1000 ml. distilled water and 3 ⁇ 600 ml. acetone.
  • the thiol gel can itself be used as an affinity chromatographic matrix or can be activated by treatment with 2,2-dithiopyridine for use with ligands containing free sulfhydryl groups.
  • the filtered thiol gel of Example 6 was suspended in 100 ml. of 20 mM dithiothreitol and stirred for 2 hours.
  • the thiol gel was washed with 3 ⁇ 1000 ml. distilled water; 3 ⁇ 1000 ml. 1 M NaCl, 3 ⁇ 600 ml. acetone and 2 ⁇ 500 ml. of a mixture of acetone and 0.05 M sodium bicarbonate containing 1 mM EDTA (40-60 v/v).
  • the washed gel was suspended in 40 ml. 0.3 M 2,2-dithiopyridine and stirred for 24-48 hours.
  • the activated thiol gel was washed successively with 3 ⁇ 600 ml. of 60% acetone; 5 ⁇ 400 ml. 1 mM EDTA and 5 ⁇ 1000 ml. 0.5 M NaCl.
  • the activated thiol gel was stored in phosphate buffered saline at 4 degrees Celsius
  • the activated thiol gel can be used, for example, to immobilize enzymes containing free sulfhydryl groups such as jack bean urease or beta-galactosidase.
  • the phenyl gel having a hydrophobic chain sterically removed from the polymeric carrier, can be used to purify proteins such as serum albumins.
  • a quantity of 45 g. of the amino-terminal gel of Example 5 was suspended in 300 ml. tetrahydrofuran. To this suspension was added 3 mmoles (0.37 g.) 4-dimethylaminopyridine; 15 mmoles (1.5 g.) succinic anhydride and 4.5 ml. triethylamine. The suspension was stirred at room temperature for 24 hours. The gel was washed successively with: 5 ⁇ 30 ml. distilled water; 2 ⁇ 600 ml. 0.5 M NaCl and 5 ⁇ 300 ml. distilled water. The gel was stored suspended in phosphate buffered saline at 4 degrees Celsius.
  • the carboxy-terminal gel can be used as an affinity chromatographic matrix for the purification of amino group containing ligands such as enzymes, antigens, antibodies, haptens, proteins, nucleoproteins, hormones and vitamins.
  • amino group containing ligands such as enzymes, antigens, antibodies, haptens, proteins, nucleoproteins, hormones and vitamins.

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dispersion Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)
  • Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
EP87310279A 1986-05-07 1987-11-20 Polymeric matrix for affinity chromatography and immobilization of ligands Expired EP0316492B1 (en)

Applications Claiming Priority (1)

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US06/860,603 US4753983A (en) 1986-05-07 1986-05-07 Polymeric matrix for affinity chromatography and immobilization of ligands

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US4885207A (en) * 1987-07-13 1989-12-05 Uop Biocompatible protein or ligand immobilization system
US4921809A (en) * 1987-09-29 1990-05-01 Findley Adhesives, Inc. Polymer coated solid matrices and use in immunoassays
US4952519A (en) * 1988-05-02 1990-08-28 E. I. Du Pont De Nemours And Company Protein immobilization with poly(ethyleneimine) derivatized with a hydroprobic group
US5028657A (en) * 1988-07-18 1991-07-02 Industrial Research Technology Institute Use of plasma to immobilize protein on polymeric surfaces
US5153166A (en) * 1988-08-18 1992-10-06 Trustees Of At Biochem Chromatographic stationary supports
CA1332598C (en) * 1989-06-20 1994-10-18 Laura J. Crane Polyethyleneimine matrixes for affinity chromatography
CS276443B6 (en) * 1989-06-30 1992-05-13 Ioannis Scarpa Spheroidal cellulose particles of high density - production method
US5130436A (en) * 1989-09-08 1992-07-14 Unisyn Fibertec Corporation 2-fluoro-1-methylpyridinium salt activated diols and polyols as cross-linkers
US6716580B2 (en) * 1990-06-11 2004-04-06 Somalogic, Inc. Method for the automated generation of nucleic acid ligands
US5232984A (en) * 1990-10-15 1993-08-03 The Board Of The Regents The University Of Texas Biocompatible microcapsules
US5380536A (en) 1990-10-15 1995-01-10 The Board Of Regents, The University Of Texas System Biocompatible microcapsules
WO1999004896A1 (en) * 1997-07-22 1999-02-04 Rapigene, Inc. Polyethylenimine-based biomolecule arrays
EP0996500A1 (en) 1997-07-22 2000-05-03 Rapigene, Inc. Apparatus and methods for arraying solution onto a solid support
US20030054360A1 (en) * 1999-01-19 2003-03-20 Larry Gold Method and apparatus for the automated generation of nucleic acid ligands
US7094351B2 (en) * 2000-10-12 2006-08-22 Corcoran Robert C Purification of substances by reaction affinity chromatography
JP3774888B2 (ja) * 2001-11-28 2006-05-17 日本化薬株式会社 ステロイド類化合物のｌｃ−ｍｓによる高感度検出法
FR2918375B1 (fr) * 2007-07-05 2009-10-16 Lab Francais Du Fractionnement Utilisation d'un support de chromatographie pour reduire la quantite d'adamts13 dans une solution derivee du plasma
JP5404620B2 (ja) * 2007-07-17 2014-02-05 ソマロジック・インコーポレーテッド 試験試料の多重化分析
CN103586009A (zh) * 2013-11-06 2014-02-19 天津大学 高密度聚乙烯亚胺修饰介质提高蛋白质吸附容量和吸附速率的方法
DE102016004432A1 (de) * 2016-04-12 2017-10-12 Sartorius Stedim Biotech Gmbh Multimodales Adsorptionsmedium mit multimodalen Liganden, Verfahren zu dessen Herstellung und dessen Verwendung
CN108212117A (zh) * 2016-12-15 2018-06-29 南京理工大学 一种三维氧化石墨烯/聚乙烯亚胺/羧甲基纤维素复合材料的制备方法
JP2020022941A (ja) * 2018-08-08 2020-02-13 日立化成株式会社 吸着材及びそれを用いた標的物質の精製方法

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4195128A (en) * 1976-05-03 1980-03-25 Bayer Aktiengesellschaft Polymeric carrier bound ligands
DE2619521A1 (de) * 1976-05-03 1977-11-24 Bayer Ag Neue aktivierte traeger und ein verfahren zu ihrer herstellung
US4415665A (en) * 1980-12-12 1983-11-15 Pharmacia Fine Chemicals Ab Method of covalently binding biologically active organic substances to polymeric substances
DE3223885A1 (de) * 1982-06-26 1983-12-29 Basf Ag, 6700 Ludwigshafen Makroporoese, hydrophile traeger fuer enzyme
US4525456A (en) * 1982-11-08 1985-06-25 Uop Inc. Support matrix and immobilized enzyme system
CA1239357A (en) * 1984-12-07 1988-07-19 That T. Ngo Method of activating polymeric carrier
JPS61191700A (ja) * 1984-12-28 1986-08-26 ジエネツクス・コ−ポレイシヨン エポキシ−ポリアルキレンイミン共重合体による生体物質の固定

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GR3003997T3 (es) 1993-03-16
ATE70539T1 (de) 1992-01-15
ES2029840T3 (es) 1992-10-01
AU8148487A (en) 1989-05-25
DE3775404D1 (de) 1992-01-30
EP0316492A1 (en) 1989-05-24
US4753983A (en) 1988-06-28
JPH01169355A (ja) 1989-07-04
JP2528486B2 (ja) 1996-08-28

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